Enteric bacilli differential media

ABSTRACT

A TEST MEDIA IS PROVIDED FOR THE POSITIVE INDENTIFICATION OF ENTERIC BACILLI BY MEANS OF DISTINCTIVE CHEMICAL AND ENZYMATIC REACTIONS.

STE/ 5 f/VCUBATE SW6 mm Aug. 27, 1974 W. ROLLENDER EHTERIC B ACILLI DIFFERENTIAL MEDIA Original Filed latch 21, 1967 STEP 1 SPECIMEN/-10 12 SJIEP 2 STEPJ" lA/CUBATE I STEP 51 B2 Mar/11W m U 2 Sheets-Sheet 1 HOURS E 0 SPECIMEN/10 522521 STEP 2 STEP-3 575. 6 [/A/cmuzj WCUBADSI 5751 7 48 Wra /ear] Wa /@2953 FIG. 2

STEP 8 STEP 9 mm SW11 wimp/2E7 $751 12 PRIOR ART mVENToRs WILL/AM EOIZENDIIZ ORV/CLEAHELKFQQD United States Patent Office Patented Aug. 27, 1974 3,832,288 ENTERIC BACILLI DIFFERENTIAL MEDIA William Rollendcr, 272 Glen Ave., Sea Cliff, N.Y. 11579, and Orville A. Beckford, 853 E. 216th St., Bronx, N.Y. 10467 Continuation of application Ser. No. 14,805, Mar. 2,

1970, which is a continuation of application Ser. No. 624,810, Mar. 21, 1967, both now abandoned. This application May 24, 1973, Ser. No. 256,328

Int. Cl. C12k 1/10 U.S. Cl. 195139 15 Claims ABSTRACT OF THE DISCLOSURE A test media is provided for the positive identification of enteric bacilli by means of distinctive chemical and enzymatic reactions.

This is a continuation of application Ser. No. 14,805, filed Mar. 2, 1970, now abandoned, which is a continuation of Ser. No. 624,810 filed Mar. 21, 1967, now abandoned.

This invention relates to test media and method for selectively identifying a particular specie of enteric bacilli.

There are many varieties of bacteria which inhabit the intestinal and urinary tract of man. Many members of the enteric group frequently found in the intestinal tract, however, do not produce any pathological disorders while others, such as Salmonella and Shigella, when found in the intestine, always produce disease. They are capable of producing dysentery, nausea, vomiting, high temperature, intestinal bleeding, invasion of the blood stream, etc.

When a doctor believes a patient is ill as a result of a bacterial infection, he orders a microbiological test. Failme to isolate and identify these organisms in the microbiology laboratory may result in serious damage to the patient. In the event inadequate treatment is given such patients, the organisms can invade internal organs causing permanent damage, internal complications and sometimes death. If the patients survive, there is the possibility that they may become Carriers, passing the infection on to others, sometimes causing epidemics.

Hospital laboratories as well as private laboratories are often faced with two major problems in this area of diagnostic microbiology; either they are staffed with inexperienced microbiology technicians or space and facilities for proper functioning are not available. As a result the majority of laboratories will take from 72 hours to one week, sometimes more, before they report the presence of a Salmonella or Shigella. During this time the patients are treated with uncertainty, and the doctor helplessly, but hopefully, awaits the reports, sometimes in vain.

The family Enterobacteriaceae comprises numerous inter-related genera all of which are microscopically undistinguishable, 3 to 5p. by 0.5 relatively straight rods with rounded ends, Gram-negative, variously motile, some are capsulated, none possess spores. All members of the family ferment glucose with or without gas production. The many common characteristics render identification of a particular specie difficult even in the hands of a skilled bacteriologist.

A new culture medium is presented hereinafter which is designed to differentiate between members of the enteric group of bacteria. The medium has the advantage of being able to differentiate between all the major groups of enteric bacteria after overnight incubation at 37 C.

Traditionally, isolation and identification of these microorganisms have been accomplished by placing the specimen onto selective and differential culture media. This culture is then placed into a 37 C. incubator, and after overnight incubation the culture media is examined for characteristic growth of bacterial colonies of the Enteric group. The colonies are then placed into differential culture media such as Triple Sugar Iron Agar or Kliger Iron Agar and again incubated at 37 C. The reactions in these differential media are based upon the organisms ability to ferment various carbohydrates and to form hydrogen sulfide. The results obtained in these media are still inconclusive, but the pattern of biochemical reactions obtained will give the microbiologist direction as to how to proceed further. Final identification of the organism may require additional culture media for further biochemical testing. Therefore, a period of more than 72 hours may elapse from receipt of the specimen to final identification of the organism. It is of utmost importance to the physician that pathogenic organisms be identified as rapidly as possible so that effective treatment of the patient can be started promptly. Public health authorities frequently must utilize such tests to track down the source of bacterial infections. The need for prompt identification to prevent the further spread of the disease is obvious.

The Enteric Bacilli Differential Medium of this invention incorporates all the salient biochemical testing into one test. In addition to differential carbohydrate fermentation, it also provides for the determination of motility, lysine decarboxylation, hydrogen sulfide production and the deamination of phenylalanine. In this fashion, one is able to differentiate Salmonella, Citrobacter, Shigella, Escheria coli and Arizona organisms. This medium will also differentiate the proteus and Providence groups as well as provide for grouping of the Proteus species. Fortyeight hours after receipt of the specimen to the laboratory, identification of the organism is accomplished. Thus, a minimum of 24 hours is saved over the prior procedure. The physician can thus begin effective treatment of the patient earlier. This media, in addition to permitting more prompt conclusive bacteriological diagnoses, also conserves the time of the microbiology technician, thereby providing increased efficiency.

Accordingly, there is need for an enteric bacterial differentiation system which permits accurate and rapid differentiation and identification, even, when carried out by inexperienced technicians.

A particular object of this invention is to provide test media having a distinct color reaction for Salmonella and Shigella.

A further object of this invention is to provide a onecontainer test system for differentiation of enteric bacteria.

These and other features, objects and advantages of the invention will, in part, be pointed out with particularity and will, in part, become obvious from the following more detailed description of the invention, taken in conjunction with the accompanying drawing, which forms an integral part thereof.

In the various figures of the drawing like reference characters designate like parts.

In the drawing:

FIG. 1 is a schematic showing of the prior art differentiation procedure accompanied by a time scale;

FIG. 2 is a schematic showing of the procedure of this invention accompanied by a time scale;

FIG. 3 is a chart showing the reactions produced by various enteric bacteria when inoculated in the prior art T.S.I. media and in the media of this invention; and

FIG. 4 is a pictorial showing of a preferred tube test chamber.

Prior art methods for identification of the enteric organisms The prior art method is shown in FIG. 1.

Step 1Time: hours The specimen 10, of feces or urine, arrives at the microbiology laboratory some form of a sterile container.

Step 2 A sample of the feces or urine is swabbed onto agar plates 12 and also put into enrichment broths. Plates commonly used include MacConkey agar, E.M.B., X.L.D. S.S., etc. Broths commonly used include G.N. Selenite and Tetrathionate. The plates are streaked starting in the swabbed area and moving out into the uninoculated portions of the plate in a pattern that creates a dilution of the sample and promotes growth of tiny isolated colonies of bacteria B B etc.

Step 3- The inoculated plates and broths are then incubated at 37 C. for 24 hours.

Step 4Time: 24 hours.

24 hours later some of the colonies that appear on the plates are picked off by means of a straight wire and inoculated into a test tube 14 containing T.S.I. agar (triple sugar iron in agar) or Kliglers iron agar.

Step 5 This is then incubated for a further 24 hours.

Step 6Time: 48 hours. The results are interpreted.

Step 7- Each T.S.I. or Kliglers tube is subjected to a series of biochemical tests, a sample from the T.S.I. tube 14 is used for testing in tubes 16a16f for the different biochemical reactions and motility test 18.

The tubes of Step 7 are incubated for 24 hours. Different biochemical patterns identify the family to which the colony belongs. Biochemical tests most commonly done are Indole, Citrate, Urea, Phenylalanine, Lysine Decarboxylase, Ornithine Decarboxylase, etc., reactions obtained from T.S.I. or Kliglers are production of gas, sugar fermentation patterns and hydrogen sulfide production. Some biochemical tests have to be incubated for a further 24 hours. Each colony must be independently investigated and all are treated independently in their future tests, e.g., colonies require 10 T.S.I. tests plus 69 tests per T.S.I. test. At this point the technician may be handling 60-100 test tubes to check the 10 colonies.

Step 9 At a minimum of 72 hours later, the bacteriologist may perform reading and interpretation of biochemical reaction set up in Step 2.

Steus 1012 The entering of the many biochemical systems in Step 7 creates a possible source of contamination resulting in erroneous reactions, thus causing confusion and the worker is unable to arrive at a conclusion after 72 hours (Step 9). Thus he is forced to return to the T.S.I. tube and again attempt to identify the organism. Thus the time range is 72 hours to one week for the prior art procedure.

4 The Rollender/Beckford method for identification of the enteric organisms The procedure of this invention is shown in FIG. 2.

Stepsl and 2Time: 0 hours The steps are the same as in the prior art. By way of example, the media used may be MacConkey agar, X.L.D. or G.N. broth. The broth is plated as in Step 1.

Step 3-Incubation.

Steps 4 and 5- After 18 to 24 hours of incubation, some of the colonies that appear on the plates 12 are picked by means of a straight wire and inoculated into the Rollender/Beckford Eenteric Differential Media. Each colony must be independently investigated by itself.

Step 6 The inoculated media is then incubated for a further 24 hours.

Step 7-Time: 48 hours 48 hours later, from the appearance of the media interpretation of important and significant biochemical reactions allow for immediate identification. The system having been incorporated in media used in Steps 4 and 5 now produces the result of these biochemical tests instead of the beginning, thus saving 24 hours.

The simplicity of the procedure employing the present invention affords the investigation of more colonies, since 10 colonies tested in the medium of this invention provides 10 answers; while 10 T.S.I. or Kliglers test would mean the setting up of approximately biochemical reactions to be read and interpreted the following day.

FIG. 3 shows, in the lower right hand corner, two tubes I and II and the prior art T.S.I. tube prior to inoculation. The other tubes show the effect of inoculation with various bacilli.

One feature of this invention is that a double pour media is employed; that is to say, there are two media, A and B, A being the one in the upper portion and B being the one in the lower portion, as shown by the differing shading for the same color in FIG. 3.

Filling is done by pouring a liquid solution into the lower portion and then permitting it to solidify. The upper portion is then poured in and permitted to solidify with the tube held at an angle to provide a slant area. It is important to have at least two butt areas.

A tube having dimensions of say mm. deep x 16 mm. in diameter may be used. About 4 cc. of solution B is poured in warm. The tube and contents are then sterilized in an autoclave. After cooling, 6 cc. of Solution A is added and permitted to solidify with the tube held at an angle.

SOLUTION A (UPPER PORTION) A. Nutrient Gms. Peptone 15.0 Yeast Extract 3.0

B. Amino Acid L-Phenylalanine 3.0 L-Tryptophan 2.0

C. Fermentation Agent Dextrose 1.0 Lactose 10.0

D. Electrolyte: Sodium Chloride 5.0

E. H 8 Indicator Ferric Ammonium Citrate 0.5 Sodium Thiosulfate 0.5

F. Solidifying Agent: Agar 15.0

G. Indicator: Brom Cresol-Purple 0.02

H. Solvent: Distilled Water, H 0-1000 cc.

SOLUTION B (LOWER PORTION) A. Nutrient Gms. Peptone 5.0 Yeast Extract 3.0

B. Lysine: Lysine 10.0

C. Fermentation Agent: Dextrose 1.0

D. Solidifying Agent: Agar 6.0

E. Indicator: Brom Cresol-Purple 0.02 F. Solvent: Distilled Water H O-1000 cc.

In Solution A the relative proportions of phenylalanine and tryptophan can be varied. The fermentation of the dextrose by certain bacteria produces acid and lowers the pH. If Brom Cresol-purple is used as the indicator, a pH of from 6.0 to 7.0 and preferably about 6.5 should be used. If another indicator is substituted, a pH suitable for the indicator should be used.

In Solutions B from 3 to 8 gms. of agar is employed to provide a semi-solid gel which permits detection of the degree of motility of the bacteria in the same media as the Lysine decarboxylation test.

To provide positive differentiation, a second tube is provided. For many years it has been known that some bacterial organisms have the capability of producing indole by the degradation of tryptophan. This has been used as a taxonomic too]. Many of the organisms we are dealing with have the ability to decarboxylate amino acids such as ornithine, and this too has been used in taxonomy. Usually a separate test must be set up to demonstrate indole production and ornithine decarboxylation. The problem in the past has been to overcome the sparing reaction of carbohydrates. Indole is not formed in the presence of carbohydrates and carbohydrates are necessary for the demonstration of ornithine decarboxylation. We have overcome this by the following formulation:

FORMULA C A. Nutrient Gms.

Peptone 20.0 Yeast Extract 3.0 B. Amino Acids L-Ornithine 5.0 L-Tryptophan 5.0 C. Fermentation Agent: Dextrose 1.0 D. solidifying Agent: Agar 15. E. Indicator: Brom Cresol-Purple 0.02

Water to make 1,000 cc.

Dissolve 49.02 grams of the above in 1,000 cc. of distilled H and boil for one minute. Dispense in 4 cc. amounts into 13 mm. X 100 mm. test tubes, stopper, and autoclave 121 C. (15 lbs. Pressure) for 15 minutes. The medium is allowed to solidify with the tube held in a slanted position so that a deep butt and a short slant is produced.

The Rollender-Beckford Enteric differential media is contained in a tube in the form of a butt and slant.

The media is inoculated with a stab wire about 3 inches in length. Having picked the desired colony, the wire is stabbed into the lower butt, then the wire is withdrawn streaking the slant at the same time. This is repeated in Tub II. The tubes are then incubated at 37 C. for 24 hours.

After incubation the ornithine decarboxylation reaction can be read from the color change in the butt portion. Positive will be purple; negative will be yellow.

Indole production is determined by removing a portion of the growth from the slant of the tube and wiping it on a paper strip IV (filter paper) that has been saturated with a solution of paradimethyl-benzaldehyde in aqueous-hydrochloric acid. If indole has been produced the paper changes to a red color. If it remains unchanged or changes to any color other than red the test is negative.

The medium may be also made by an alternative formula using 5 gms. of peptone instead of 20. This media could also be made in a double pour like the first one 6 where the ornithine decarboxylase medium would be on the bottom and the indole sensing medium on top; that is to say, the composition of Formula C minus the ornithine would be on the top, and Formula C minus the tryptophan on the bottom.

In FIG. 3 there is shown a chart represented by test tubes containing the test media of this invention. Tube I contains mediums A and B and Tube II contains medium C. In its uninoculated form the tubes have a purple color. Shown with the tube of this invention is the standard T.S.I. tube. After 24 hours incubation following inoculation with the selected colony of bacteria, various reactions occur, as shown on the chart. It will be noted that the prior art T.S.I. may react in at least four different ways, none of which specifically indicate the bacteria present, and, in fact, may merely indicate the presence of any one of from two to seven species of bacteria. On the other hand, it will be seen that with the media of the present invention there is a clear differentiation of most bacteria, the only similarities occurring being in the case of E. coli and Serratia, Hafnia and Aerobacter. A simple paper spot test IV will immediately differentiate the E. coli. There is another common reaction between P. rettgeri and Providence species but which creates no problem as a positive identification can be made by the standard urea test. On the other hand, there i a group of seven species of bacteria including the pathogenic Shigella specie which produce identical T.S.I. reactions.

Salmonella organisms isolated in this manner give a distinct purple slant, black band upper butt and purple lower butt.

Shigella organisms isolated in this manner give a distinct purple slant yellow upper butt and yellow lower butt, no gas produced.

Citrobacter organisms which formerly presented difiiculty in differentiation and was a great source of confusion with the Salmonella species, now clearly distinguishes itself with a purple slant, black band in the yellow upper butt and yellow lower butt (no exceptions).

Proteus vulgaris and Proteus mirabilis isolated in this manner gives a distinct red-brown slant, black band in the yellow upper butt and a yellow lower butt.

Proteus morganii, Proteus rettgeri and the Providence group gives a distinct red-brown slant and yellow upper butt and yellow lower butt.

E. coli and Aerobacter produces various forms of yellow slant and yellow butt with the gas produced, causing separation. All color combinations are typical for the biochemical and fermentation reactions for which these organisms were formerly grouped and identified; because of the combinations used there are no false positives and these color reactions are specific for the groups they represent.

In tube I the black band and/or black upper butt results from the formation of H 5.

The lysine decarboxylation reaction can be read from the color change in the butt portion. Positive will be indicated by purple; negative will be indicated by yellow. Motility can be ascertained by the degree of diffusion of the organisms throughout the medium. Deamination of phenylalanine is evidenced by a change in the indicator in the slant portion of the upper medium, a distinctive red-brown color being produced.

In place of two separate tubes I and II, it is preferred to have a double container such as shown in FIG. 4.

The container is in the form of a transparent bottle 50 either glass or sterilizable plastic, fitted with a screw cap 52. A barrier 51 separates the interior into two compartments one of which contains media A and B and the other media C.

The advantage of this container is that the microbiologist can unscrew the cap by capturing the cap in the crook of the little finger or between his index finger and middle finger and twisting the bottle, then with the wire held between the thumb and finger he can introduce the specimen through a common area for the two chambers. The screw caps are most convenient and provide at low cost a good seal.

Having thus disclosed the best embodiment of the invention presently contemplated, it is to be understood that various changes and modifications may be made by those skilled in the art without departing from the spirit of the invention.

What we claim as new and desire to secure by Letters Patent is:

1. An enteric bacilli differentiation apparatus containing three separate media in combination comprising:

I. A first medium and a second medium (a) said first medium comprising a gel non-toxic to the bacilli to be dilferentiated having dispersed therein;

(1) Sufiicient nutrient to support the growth of bacilli to be differentiated;

(2) Sufiicient amino acid and a sufficient source of ferric ions to provide an indication of deamination of the amino acid by selected bacilli;

(3) Sufficient carbohydrate to provide an indication of fermentation and to act as a catalyst for decarboxylation;

(4) A source of sulphur for enhancing production of H 8 in the presence of selected bacilli;

(5) A pH indicator;

(b) said second medium comprising a gel containing lysine and including means for the detection of the decarboxylation of the lysine in the presence of bacilli causing the decarboxylation of the lysine; and

(6) At least one transparent container containing said first and second mediums, said first medium overlying said second medium.

II. A second transparent container containing a third medium comprising means for the detection of indole produced by selected bacilli.

2. The apparatus of Claim 1 wherein the surface of said first medium is sloped.

3. The apparatus of Claim 1 wherein said second medium includes:

(a) sufiicient nutrient to encourage the growth of bacilli to be differentiated; and

(b) a carbohydrate.

4. The apparatus of Claim 1 wherein said first and said second transparent envelopes are in a common envelope.

5. The apparatus of Claim 1 wherein said indicator is Brom Cresol-Purple.

6. The apparatus of Claim 1 wherein said third medium is a gel which contains:

(a) a nutrient for the bacilli to be diiferentiated;

' (b) sufiicient L-Ornithine to provide an indication of decarboxylation by selected bacilli;

(c) sufiicient tryptophane to provide indole by degradation by selected bacilli; and

(d) carbohydrate.

7. An enteric bacilli differentiation apparatus containing two separate media in combination comprising:

I. A first transparent container containing a first medium and a second medium whose surface is completely covered by said first medium;

(a) said first medium comprising a gel nontoxic to the bacilli to be differentiated having dispersed therein;

(1) Sufficient nutrient to support the growth of bacilli to be differentiated;

(2) Sufficient amino acid and a suflicient source of ferric ions to provide an indication of deamination of the amino acid by selected bacilli;

8 (3) Suflicient carbohydrate to provide an indication of fermentation and to act as a catalyist for decarboxylation; (4) A source of sulphur for enhancing production of H 8 in the presence of elected bacilli; and (5) A pH indicator; and (b) said second medium comprising a gel containing lysine and including means for the detection of the decarboxylation of the lysine in the presence of bacilli causing the decarboxylation of the lysine. 8. The apparatus of Claim 7 wherein the surface of said first mediumis sloped.

9. An enteric bacilli differentiation apparatus containing three separate media in combination comprising:

(a) a first medium comprising:

Parts Petone 15.0 Yeast Extract 3.0 Phenylalanine 3.0 Tryptophan 2.0 Dextrose 1.0 Lactose 10.0 Sodium Chloride 5.0 Ferric Ammonium Citrate 0.5 Sodium Thiosulfate 0.5 Agar 15.0 Brom Cresol-Purple 0.2 Water 1000 (b) a second medium comprising:

Parts Peptone 5.0 Yeast Extract 3.0 Lysine 10.0 Dextrose 1.0 Agar 6.0 Brom Cresol-Purple 0.02 Water 1000 (c) a third medium comprising:

Parts Peptone 20.0 Yeast Extract 3.0 L-Ornithine 5.0 Tryptophan 5.0 Dextrose 1.0 Agar 15.0 Brom Cresol-Purple 0.02 Water 1000 10. The apparatus of Claim 9 wherein said second medium is a semi-solid gel.

11. The apparatus of Claim 9 including a transparent container having said first and said second medium in abutting layers.

12. The apparatus of Claim 11 wherein said first media is above said second medium.

13. The apparatus of Claim 12 wherein said container is elongated along a principal axis and said first medium is solid and has a slant surface in a plane at an acute angle to the principal axis.

14. The apparatus of Claim 11 wherein said transparent container has an internal divider forming two compartment and said first and said second medium are in one said compartment and the other said medium is in another of said compartments.

15. An enteric bacilli differentiation apparatus comprising in combination:

(a) a first transparent container and a first medium contained therein said first meduim comprising:

Peptone15.0 partsiLO percent Yeast Extract3.0 parts:2.0 percent Phenylalanine3.0 parts:0.5 percent Tryptophan2.0 parts:0.5 percent 'Dextrose1.0 parts-$0.5 percent 9 10 Lactose10.0 partsiLO percent where the percent is parts by weight of total composition. Sodium Ch1oride5.0 partsiLO percent Ferric Ammonium Citrate0.5 'partsi-0.5 percent References Cited Sodium Throsu1fate0.5 parts: 1.5 percent UNITED STATES PATENTS Agar-15.0 parts-$1.0 percent 1' Brom Creso1 Purple-0.2 parts-$0.001 percent 2992974 7/1961 Belcone 195 139 partsio Percent 3,205,151 9/1965 Landaw et a1. l95-103.5 R 3,233,974 2/1966 Bradley 23-253 (b) a second rnedrurn contained 111 said container said 3 7 3 4 19 3 Watson at 2 230 Second medium compflslngl 3,278,393 10/1966 Bahn et a1 195100 Peptone5.0 parts:1.0 percent 10 3,234 ;107 2/1966 Kaufman et a1 195139 {e353 Extlrggt-jf Eggs- 2.0 ptercent O R REFERENCES e cen pgs g Fgs1e9Be11%t;er101jo%19c4a1 Culture Med1a, 1967 Fisher SCL, Agar-6.0 partsi0.3 percent f: b i l M h E 1 Brorn Creso1Purple-0.2 parts: 0.001 percent t 1 {a e for t e mmbmema D1 0 parts+0 percent Labs., Detrolt, M1ch., pp. 3-7. Singer et al., Journal of Bacteriology, 69: 303, 1955. a seflolld pfl colllalller f d a thifd ll ledlum Benson-Microbiological App1ic.-W. C. Brown & Co.,

contained therein said third med um comprising: Publisher, p. 96, 1967.

Peptone20.0 partsiLO percent Yeast Extract 30 partsizo-percem A. LOUIS MONACELL, Primary Examiner L-Ornithine5.0 parts-$1.0 percent R. I. WARDEN, Assistant Examiner Tryptophan5.0 partsiLO percent Dextrose1.0 parts:0.5 percent -U.S. C1. X.R.

Agar-45.0 partsi0.5 percent -100, 103.5 R

Brorn Creso1 Purple0.02 partsiflOfll percent Water-1000 partsiO percent 

